Actin assembly and disassembly at the plasma membrane generate dynamic movements to control fundamental cell processes. Studies with gelsolin null cells shown that gelsolin is essential for membrane ruffling, a process driven by uncapping/nucleated actin polymerization at the cell periphery. In vitro studies show that gelsolin is activated by micromolar Ca2+ to sever, and is dissociated from actin nuclei (uncapping)by polymphosphoinositide 4,5-bisphosphate (PIP2). Gelsolin's severing and capping activities are potentiated by profilin, which delivers actin monomers to the uncapped actin nuclei. These findings are incorporated into an uncapping/nucleated actin assembly model. Our plan is to understand how gelsolin is regulated by Ca2+ and PIP2, by characterizing their binding and functional effects with a combination of biochemical, molecular and structural approaches. We will subsequently apply the information obtained by in vitro experiments to the in vivo arena. First, determine the structural basis for Ca2+ regulation of gelsolin. We have obtained a X-ray crystal structure of gelsolin complexed with actin in Ca2+ and propose a mechanistic model for how Ca2+ binding leads to actin binding. Aspects of this model will be tested. Second, examine the structural basis for PIP2 binding by gelsolin. We will examine how Ca2+ increases PIP2 binding affinity, and will characterize a newly identified PIP2 binding site in the gelsolin C-half. PIP2 binding site residues in the and C-halves will be mutated to generate PIP2 regulation mutants. Third, use gelsolin loss of function mutants to identify the requirements for gelsolin-mediated ruffling in live cells. Gelsolin null cells will e used as living test tubes for in vivo reconstitution with gelsolin functional or regulatory mutants. Rescue of ruffling will identify gelsolin functions that are necessary and sufficient for dynamic actin assembly. Fourth, develop the gelsolin cameleon probe to monitor gelsolin activity in live cells. This probe will allow us to visualize gelsolin interactions with actin in vivo, a measurement which cannot be obtained directly by any other existing single cell based microscopic method. We will use this new tool to test the uncapping/nucleated actin assembly model. The combined approaches in this grant will help elucidate how actin dynamics are regulated at the plasma membrane and will have relevance to many membrane events which are regulated by the crosstalk between membrane signaling and cytoskeletal remodeling.